Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82

Abstract

Massively parallel sequencing of targeted regions, exomes, and complete genomes has begun to dramatically increase the pace of discovery of genes responsible for human disorders. Here we describe how exome sequencing in conjunction with homozygosity mapping led to rapid identification of the causative allele for nonsyndromic hearing loss DFNB82 in a consanguineous Palestinian family. After filtering out worldwide and population-specific polymorphisms from the whole exome sequence, only a single deleterious mutation remained in the homozygous region linked to DFNB82. The nonsense mutation leads to an early truncation of the G protein signaling modulator GPSM2, a protein that is essential for maintenance of cell polarity and spindle orientation. In the mouse inner ear, GPSM2 is localized to apical surfaces of hair cells and supporting cells and is most highly expressed during embryonic development. Identification of GPSM2 as essential to the development of normal hearing suggests dysregulation of cell polarity as a mechanism underlying hearing loss.

Figure 1

GPSM2 and Nonsyndromic Hearing Loss DFNB82 in Palestinian Family CG (A) Family CG with inheritance of GPSM2 p.R127X and hearing loss (filled symbols). (B) Hearing loss in members of family CG. The affected individuals are ages 12 (CG11), 14 (CG10), 21 (CG6), and 24 (CG7). For CG20, age 2, brainstem-evoked response tests showed no response for stimuli at 90 dB in either ear. (C) Verification by Sanger sequencing of nonsense mutation c.875C>T (p.R127X) in GPSM2 (accession number NM_013296). (D) Site on the GPSM2 protein of the p.R127X truncation (arrow). T1 to T7 indicate tetratricopeptide repeats; G1 to G4 indicate GoLoco motifs. The project was approved by the Human Subjects Committee of Bethlehem University and by the Human Subjects Division of the University of Washington.

Figure 2

Localization of Gpsm2 in the Mouse Inner Ear (A and B) Schematic representation of the cochlea (A) and utricle (B), one of the vestibular organs. The inner and outer hair cells of the cochlea make up the sensory cells; they are embedded in nonsensory supporting cells, including the Deiters, pillar, and Hensen cells. The utricle also contains hair cells and supporting cells. (C–J) Immunofluorescence images of the cochlea at e16.5 (C and D) and p0 (G and H) and of the utricle at e16.5 (F) and p0 (J). Gpsm2 (green) is localized at the apical surface of the hair and supporting cells, both in the cochlea and utricle and along the greater epithelial ridge (E and I) in e16.5 and p0 inner ears. In p0 cochlea, it is specifically localized at the lateral ridge of the hair cell surface. It is also localized in the pillar cells at this age. Hair cell cytoplasm is marked by myosin VI (red), and the nuclei are marked by DAPI (blue). Paraffin embedded sections were stained with primary antibodies goat anti-myosin VI (Santa Cruz Biotechnology) and rabbit anti-Gpsm2 (ProteinTech; for validation of the antibody, see 12) and the fluorescence-conjugated secondary antibodies donkey anti-rabbit 488 (Molecular Probes) and donkey anti-goat cy3 (Sigma). Imaging was done with an LSM 510 confocal microscope (Zeiss). Scale bars represent 5 μm (C, E–G, I, and J), 2 μm (D), and 2.5 μm (H). All procedures involving animals met NIH guidelines and were approved by the Animal Care and Use Committees of Tel Aviv University and the University of Washington.

Figure 3

Expression of Gpsm2 in the Mouse Inner Ear during Development Expression of Gpsm2 transcript at ages e16.5, p0, 1 week, 1 month, and 3 months, determined by quantitative real-time PCR (Applied Biosystems, TaqMan assay Mm00512842). Values represent mean ± standard deviation of samples run in triplicate. The experiment was repeated twice; a representative experiment is shown.